The present invention relates to an optical assembly comprising a sample vessel positioned in a direct light path between a light source and a light detector, in manner to enable transmission of light through the vessel; a method for detection of light transmission through sample contained within the vessel; an apparatus comprising the assembly; more particularly an apparatus for sample analysis for example for high throughput screening (HTS) or profiling or assays, such as enzyme assays; and uses thereof in the pharmaceutical, biomedical and bioscience, agrochemical, veterinary, materials and like fields, for detection, analysis, characterization and quantification or the like of samples contained in a vessel, and optionally further collecting separated components thereof; in particular in combinatorial chemistry; in metabolomics, proteomics or genomics, assay and high throughput analysis applications, typically high sensitivity analyses, separation and/or quantification studies and for sample separation for example chromatography or electrophoresis, in particular column chromatography, capillary electrophoresis with real time or post separation analysis.
UV absorbance, fluorescence and mass spectrometry are key technologies used in separation science for analyzing species in samples. A particularly useful methodology is to look at a sample population separated by capillary electrophoresis with fluorophore labelling and fluorescence imaging, for quantification, and MS for characterizing molecules of interest.
U.S. Pat. No. 5,582,705 discloses an apparatus and system for laser induced fluorescence (LIF) detection in a multiplexed capillary electrophoresis system. A coherent beam incident on the capillary array and emitted fluorescent light are typically perpendicular to each other in order to reduce background noise due to light scattering. A transparent portion in each capillary wall defines a transparent path extending through the array, perpendicular to the capillary. A 2D image array detector such as a charge-coupled device (CCD), preferably a charge-injection device (CID), is positioned to detect emission, and an imaging lens interposed between the capillary array and the image array detector, to optically couple the pixels to the capillary. The imaging lens may be any lens capable of transforming an image onto the pixels of the image array detector, such as a camera lens or a condenser lens. Coupling is shown in FIG. 4, of U.S. Pat. No. 5,582,705 in which every second pixel is coupled to a sidewall of the capillary and every pixel in between is coupled to an interior portion.
Fluorescence detection is limited in its application since only a limited number of molecules are naturally fluorescent and many have to be derivatized in reproducible and quantitative manner. Absorbance detection therefore has the advantage of enabling detection of a wider range of molecules. For example in enzyme assays, conducted in microtitre wells, techniques can be extended to absorbance detection of chromophoric, UV and vis absorbing substrates consumed or produced in an assay, extending the range of assay to natural as well as synthetic substrates.
However a limitation of absorbance detection lies in the operable wavelength of detection. Absorbance detection is conducted on substrates in solution. However many common solvents absorb significant amounts of light at wavelengths below ˜200 nm, and the resulting solvent absorption signal distorts and masks signals resulting from the substrate to be detected. Accordingly absorbance detection is in practice limited to detection at wavelengths in excess of 190 nm, in the range UV-vis to near infra-red (NIR).
Moreover a fundamental limitation of single point absorbance detection is the impossibility of creating an image of the source at the detection point on capillary that is brighter than the light source. In “A charge coupled device array detector for single-wavelength and multi-wavelength ultraviolet absorbance in capillary electrophoresis”, Bergstrom and Goodall, Pokric and Allinson, Anal. Chem. 1999, 71, 4376-4384 discloses optical detection in capillary electrophoresis by means of absorbance detection, illuminating a length of the capillary using a fiber optic bundle and using a charge coupled device (CCD) camera to image the full length of the illuminated zone. In this publication light from a fiber optic bundle is focused by a sapphire rod through the capillary core and detected on the opposite side of the capillary, by this means, increasing the target light area enabling more of the lamp output to be used and increasing the total light flux. In this case light emanates from the capillary core, so all light detected is useful and the divergent beam obtained is imaged on to the CCD.
Such a system becomes more complex once a parallel capillary array is introduced in place of the single capillary. Optics to focus light on the core of each capillary would be extremely complex and therefore irradiating both the core and walls of each capillary becomes a practical consequence.
WO 01/18528 (Yeung et al) discloses a method for analyzing multiple samples simultaneously by absorption detection of samples in a planar array of multiple containers, whereby stray light from adjacent containers is eliminated by distancing the detection means from the array, preferably at a distance greater than 10 times the diameter of a container, suitable 10-100 times the diameter for example at a distance of 1-30 cm. Containers are preferably cylindrical capillary tubes as shown in the art. The array comprises a control container if the light source is unstable. It is stated that the cross section of the container and thickness of the capillary wall are not critical. A flat field lens preferably images the containers on to the detection means.
We have now found that further improvements in absorbance detection assemblies enables increasing the total light flux through a capillary or other sample vessel, by virtue of simplification of optical components, without unduly large separation of capillary and detector which is undesirable and reduces light collection efficiency compromising path length, and therefore light intensity. The improved assembly is of particular advantage in detection in multiplexed capillary arrays and enables imaging a large area of a capillary array without the need for imaging optics. This is a significant advantage, especially when working in UV for which it is very difficult and expensive to produce suitable optics. The assembly has benefits however in both single capillary and array detection, in particular enabling a simple and improved exposure referencing and acceptably low intercapillary cross talk without the need for optics. In addition a benefit of the assembly of the invention is that it is suitable for operation at short pathlengths, by virtue of the increased total light flux through the core of the capillary or other sample vessel, and this reduction in pathlength may lead to opportunities to conduct absorbance detection at lower wavelengths, less than 190 nm, without encountering impracticably high levels of solvent absorption.